Tài liệu Báo cáo khoa học: Regulatory modes of rod outer segment membrane guanylate cyclase differ

Regulatory modes of rod outer segment membrane guanylate cyclase

differ in catalytic efficiency and Ca2+-sensitivity

Ji-Young Hwang1,*, Christian Lange1,†, Andreas Helten1

, Doris Ho¨ ppner-Heitmann1

, Teresa Duda2

,

Rameshwar K. Sharma2 and Karl-Wilhelm Koch1

1

Institut fu¨r Biologische Informationsverarbeitung 1, Forschungszentrum Ju¨lich, Ju¨lich, Germany; 2

The Unit of Regulatory and

Molecular Biology, Departments of Cell Biology andOphthalmology, NJMS & SOM, UMDNJ, Stratford, NJ, USA

In rod phototransduction, cyclic GMP synthesis by mem￾brane bound guanylate cyclase ROS-GC1 is under Ca2+-

dependent negative feedback control mediated by guanylate

cyclase-activating proteins, GCAP-1 and GCAP-2. The

cellular concentration of GCAP-1 and GCAP-2 approxi￾mately sums to the cellular concentration of a functional

ROS-GC1 dimer. Both GCAPs increase the catalytic effi￾ciency (kcat/Km) of ROS-GC1. However, the presence of a

myristoyl group in GCAP-1 has a strong impact on the

regulation of ROS-GC1, this is in contrast to GCAP-2.

Catalytic efficiency of ROS-GC1 increases 25-fold when it is

reconstituted with myristoylated GCAP-1, but only by a

factor of 3.4 with nonmyristoylated GCAP-1. In contrast to

GCAP1, myristoylation of GCAP-2 has only a minor effect

on kcat/Km. The increase with both myristoylated and non￾myristoylated GCAP-2 is 10 to 13-fold. GCAPs also confer

different Ca2+-sensitivities to ROS-GC1. Activation of the

cyclase by GCAP-1 is half-maximal at 707 nM free [Ca2+],

while that by GCAP-2 is at 100 nM. The findings show that

differences in catalytic efficiency and Ca2+-sensitivity of

ROS-GC1 are conferred by GCAP-1 and GCAP-2. The

results further indicate the concerted operation of two

GCAP modes that would extend the dynamic range of

cyclase regulation within the physiological range of free

cytoplasmic Ca2+ in photoreceptor cells.

Keywords: phototransduction; guanylate cyclase; GCAP;

myristoylation; kcat/Km.

Photoexcitation of vertebrate photoreceptor cells leads to

the hydrolysis of cyclic GMP (cGMP) and subsequent

closure of the cyclic nucleotide-gated (CNG) channels in the

plasma membrane. Restoration of the dark state of the

photoreceptor cell requires the reopening of CNG-channels

(reviewed in [1–3]). A critical step in this recovery process is

synthesis of the second messenger, cGMP. Studies with

vertebrate photoreceptor cells, constituting mainly rods,

show that these cells express two types of a membrane

bound guanylate cyclase termed ROS-GC1 and ROS-GC2

(alternatively used names are retGC1 and retGC2 and

GC-E and GC-F; reviewed in [4,5]). ROS-GC1 has been

purified directly from bovine and amphibian rod outer

segments [6–9], and it is the only cyclase which has been

cloned based on its amino acid sequence [8,10]. Human

retinal diseases (LCA1 and CORD6) affect both rod and

cone vision, but are only linked to the ROS-GC1 gene

[11–17]. Knowledge about enzyme kinetic parameters of

native photoreceptor guanylate cyclase are so far restricted

to ROS-GC1. This is mainly because only ROS-GC1 has

been purified from bovine retina and thus, probably,

constitutes the main cyclase in bovine rod outer segment

preparations. Reported Km-values for the substrate, GTP,

range from 0.76–1.1 mM [6,7,9,18]. Turnover numbers (kcat)

of the purified enzyme range from 0.2–3.9 cGMPÆs

)1 [6,9].

Small acidic Ca2+-binding proteins, called guanylate

cyclase-activating proteins or GCAPs, regulate ROS-GC1.

Three GCAP (GCAP-1, 2 and 3) isoforms have been cloned

from retinal sources [19–23]. GCAP-1 and GCAP-2 are

both expressed in rod and cone cells of different species as

shown by immunocytochemistry [21,22,24,25]. Expression

of GCAP-3 is more restricted; it is present in human cones,

fish rods and cones, but not in mice photoreceptor cells [26].

Thus, GCAP-3 does not appear to be a general sensor of

Ca2+-pulses linked with phototransduction.

GCAP-1 and GCAP-2 contain one nonfunctional and

three functional EF-hands. Through functional hands they

detect changes in the intracellular Ca2+-concentration

[Ca2+] and modulate ROS-GC1. Dark adapted vertebrate

photoreceptor cells have a cytoplasmic free [Ca2+] of 500–

650 nM. This falls below 100 nM upon illumination [27–30].

GCAPs detect the fall and in their Ca2+-free form, activate

ROS-GC1 [4,5,19–23]. The generated cyclic GMP replen￾ishes the depleted pool and restores the channels in their

open state. While there is wide agreement in the literature

Correspondence to K-W. Koch, Institut fu¨r Biologische Information￾sverarbeitung 1, Leo-Brandt-Strasse, Forschungszentrum Ju¨lich,

D-52425 Ju¨lich, Germany.

Fax: + 49 2461 614216, Tel.: + 49 2461 61-3255,

E-mail: [email protected]

Abbreviations: ROS, rod outer segments; ROS-GC1/GC2, photo￾receptor membrane guanylate cyclases 1 or 2; GCAP-1/2, guanylate

cyclase activating protein 1 or 2; NMT, N-terminal myristoyl trans￾ferase; myr, myristoylated; nonmyr, nonmyristoylated; Rh, rhodopsin.

Enzymes: guanylate cyclase (EC 4.6.1.2.)

*Present address: Genetics & Molecular Biology Branch National

Human Genome Research Institute National Institute of Health Bldg.

49, Rm 4A08, 49 Convent Drive, Bethesda, MD 20892–4442, USA.

Present address: Instituto de Bioquı´mica Vegetal y Fotosı´ntesis,

Centro de Investigaciones Isla de la Cartuja, Avda. Ame´rico Vespucio

s/n, 41092 Sevilla, Spain.

(Received 2 June 2003, accepted 28 July 2003)

Eur. J. Biochem. 270, 3814–3821 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03770.x